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Testicular Histology in Cryptorchid Boys – Aspects of Fertility

Background: Cryptorchidism is associated with infertility. The aim of this study was to evaluate the progression of germ cell damage during childhood of cryptorchid boys.

Material and methods: The material included 963 cryptorchid patients <12 years at surgery with biopsy at surgery (1168 specimens) and testicular tissue from 35 human cryptorchid fetuses >28 weeks of gestation with intra-abdominal or intracanalicular tests (70 specimens). From the testicular biopsies the number of spermatogonia and gonocytes, if any/tubule was measured and compared to a normal material.

Results: All cryptorchid fetuses exhibited germ cells. Germ cells disappeared from 18-months of age. Hereafter, the frequency of no germ cells at surgery increased with increasing age (p<0.00001). In 77% (27/35) of the cryptorchid fetuses a normal number of germ cells were found. The number of germ cells decreased dramatically within the first two years of life. After 2 years of life the frequency of a normal number of germ cells was about 9% only.

Conclusions: We recommend surgery for cryptorchidism before the germ cells disappear, i.e. before 15-months of age; especially in bilateral cryptorchidism. We recommend a testicular biopsy simultaneously with surgery for cryptorchidism. The histological findings may be helpful when deciding whether a cryptorchid boy older than 15 months may be offered supplementary hormonal treatment in order to stimulate germ cell proliferation after surgery.

Cryptorchidism is associated with infertility. Early surgery is recommended in order to preserve testicular germ cell maturation (1). At orchiopexy testicular biopsies may be indicated to evaluate the fertility potential (1-4). In cryptorchidism the mean number of spermatogonia and gonocytes per tubular cross section (mean-S/T) in percent of the age-specific value correlates positively with the sperm count in adulthood (2-4). When the S/T-values bilaterally are normal in biopsies taken at orchiopexy for bilateral cryptorchidism at 10- 16 years of age, a normal sperm count is found in adulthood. If decreased mean S/T-values are found, the risk of later infertility is high and related to the S/T-values (4).

During the first months of life the hypothalamic-pituitarygonadal hormone axis is transiently activated resulting in increased serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone and inhibin B. The elevated inhibin B persists for a longer period of time than the elevated FSH, LH and testosterone, as it remains elevated to 15 months of age and decreases to basal level during the third year of life (5, 6, and 7). During the prepubertal period inhibin B is secreted in detectable amounts, while FSH, LH and testosterone are extremely low (8). During puberty inhibin B becomes inverted to FSH (6, 8, and 9). The changes in the FSH, LH and testosterone-values reflect the histological changes in the testes. The total number of germ cells and the number of Leydig cells reach a peak value around the 3rd month of life (3,10), and the main proliferative activity of the Sertoli cells takes place during the first 2-3 months of life (3). The transformation from gonocytes to the adult reservoir of germ stem cells, the Ad Spermatogonia starts in the third trimester, to be completed after the 6th month of life (1, 10). In cryptorchidism the transformation is defective, and the gonocytes may persist until about 1.5 years of age. In cases of no gonocyte transformation, no Spermatogonia A dark appear, and later on germ cells lack in testicular biopsies (1).

The germ cell proliferation and transformation may also later in childhood be hormonal dependent (1, 10, and 11). Treatment with a luteinizing hormone-releasing analogue after successful surgery for cryptorchidism in childhood markedly improved the sperm counts later in life (11). However, hormonal treatment may harm the germ cells too, and unsuccessful hormonal treatment of cryptorchidism has been reported to reduce the number of germ cells (12, 13). It is therefore important to achieve knowledge about the general progression of germ cell damage during childhood of the cryptorchid boys.

Material and methods

The material included 963 consecutive cryptorchid boys younger than 12 years who had surgery for cryptorchidism with simultaneous successful testicular biopsy, between Januar 1971 and March 2006. Excluded were patients who had undergone prior inguinal surgery or exhibited retractile testes, those with a uterus, tuba uterina, testicular neoplasia or known X chromosome, and those with biopsies not demonstrating testicular parenchyma (3, 4, 14). There were 305 bilateral and 658 unilateral cryptorchid boys. In 67% (205/305) of the bilateral cryptorchid boys bilateral biopsies demonstrating testicular parenchyma were present. The median (range) age at surgery was 7.0 (0.1-11.9) years for the bilateral and 6.1 (0.03-11.9) years for the unilateral cryptorchid boys. Totally 95/963 patients (10%) of the patients had intra-abdominal testes. Among the 658 unilateral cryptorchid patients 54 (8%) had an intra-abdominal testis.

Among the 305 bilateral cryptorchid patients: 41 patients (13%) had intra-abdominal testes: 21 patients had bilateral intra-abdominal testes, 15 patients had one intra-abdominal testis and one undescended testis distal for the abdominal cavity, and 5 patients had one intra-abdominal testis and lacked the contralateral testis. Furthermore, the material consisted of 35 cryptorchid fetuses that died between gestational week 28 and 40. The fetuses were cryptorchid as the testes were located in an intraabdominal or intra-canalicular position. From 28 weeks of gestation the testes are normally descended beyond the external inguinal ring (3). The fetuses were stillborn or died within the first 3 days of life, and had undergone autopsy, including fixation of the testes. In 10 cases a normal 46, XY karyotype was proved, and the remaining cases presented with a normal 46, XY phenotype (15).

The testicular tissue specimens were fixed in Stieve´s solution, embedded in paraffin, and 4-μm sections were at least stained with hematoxylin-eosin. The number of spermatogonia and, gonocytes if any, per tubular crosssection (S/T) was assessed based on at least 100 tubular crosssections (3). For every patient, the age-matched S/T was calculated. This value appeared as the assessed S/T divided with the lowest normal S/T for the age, in accordance to our published normal values (3, 15). The S/T was considered normal when the value was at least 0.69 in 9 months to 1 year old boys, at least 0.38 in 1 to 4 years old boys, at least 0.65 in 4 to 10 years old boys and at least 1.80 in 10 to 12 years old boys. It was noted if the biopsy of the cryptorchid boys lacked germ cells, including biopsies showing less than 1% of the lowest normal S/T-value for the age. Every patient appears once, and consequently in bilateral cases the mean value from the two testes was used. The study was performed in agreement with the Helsingki II declaration and was approved by the local ethics committee. For statistical analysis, the Fischer´s exact and Chi-square test were performed. Two-tailed tests were done and p<0.05 was considered significant.

Results

The risk of no germ cells in cryptorchidism: Germ cells were demonstrated in all cryptorchid fetuses. The youngest cryptorchid patient without germ cells was 18 months of age. The frequency of no germ cells was increasing with increasing age (Chi-square test, p<0.00001) (fig. 1). Totally, 20% (196/963) of the patients lacked germ cells. Germ cells lacked in 19% (123/658) of the unilateral cases, which was possibly less compared to the 24% (73/305) of bilateral cryptorchid boys who lacked germ cells in one or both testes (Chi-square test, p = 0.0833). Germ cells lacked bilaterally in 75% (55/73) of the bilateral cryptorchid patients who lacked germ cells.

The frequency of a normal number of germ cells at surgery: In 77% (27/35) of the cryptorchid fetuses a normal number of germ cells were found. The number of germ cells decreased dramatically within the first two years of life (fig. 2). After 2 years of life the frequency of a normal number of germ cells was about 9% only (fig. 3).

Discussion

Germ cells were present in all cryptorchid fetuses. During the first 2 years of age the number of germ cells dramatically decreased. The youngest cryptorchid patient without germ cells in the biopsy was 18 months of age. Equivalently, the literature reports lack of germ cells from cryptorchid boys aged 15 (16) and 18 months (17). After this age a progressive number of patients lacked germ cells at surgery for cryptorchidism, even in unilateral undescended testes. Normally, the primary spermatocytes appear at 4 years of age and spermatogenesis stops at this level until about the 12th year of life (3, 18, and 19). The undescended testes are not quiescent organs during childhood, as a progressive loss of germ cells takes place. Intra-abdominal testes had the highest risk of no germ cells, as reported earlier (19). It may reflect that the temperature is higher intra-abdominally than at the superficial inguinal pouch.

The values from undescended testes were compared to our published normal values. From a scientific point of view more specimens from otherwise normal boys would have been optimal, but from an ethical point of view it was not possible. This is the general problem in obtaining normal values in children, and our normal values are not conflicting previous studies (2, 3, 10, 18, and 19).

It is important that lack of germ cells in biopsies at surgery is associated with subsequent infertility. In bilateral cryptorchidism the risk of infertility is 78% to 100% dependent on if germ cells lacked in one or both testes (4). In unilateral cryptorchidism the risk of later infertility is 33% ( 4). The number of germ cells at surgery is important for the subsequent fertility potential (2, 18, 20), but is notable that lack of germ cells may result in infertility even in unilateral cryptorchidism. This may be because the same pathological mechanisms are operating in both testes. Our results emphasize the importance of performing orchiopexy before 15 month of age, because after that age the testis may already lack germ cells. However, early surgery may not be sufficient in all cases in order to preserve the fertility potential. When infertility occurs in formerly unilateral cryptorchid patients, the risk of a relative deficiency of FSH is very high. A mild form of hypogonadotropic hypogonadism has been reported in cryptorchid boys (1, 10, 11), and insufficient LH- and FSHresponse to a GnRH-stimulating test was reported in otherwise normal cryptorchid boys (21). In general, deficiency of FSH was suspected in 21% of patients with former bilateral and in 8% in unilateral cases (14). Consequently, surgical treatment may not be sufficient in all cases of cryptorchidism, but supplementary hormonal treatment may be indicated after surgery (11).

Recently it has been shown hCG treatment induced a significant increase in plasma testosterone required for Ad spermatogonia development, even up to 7 years of age (22). However, a reduced number of germ cells have been reported in 1 to 3-year old cryptorchid boys after unsuccessful hormonal treatment with gonadotropin releasing hormone or hCG (13), and in 10 months to -13 years old cryptorchid boys after unsuccessful treatment with hCG (12). So the results of the studies in the literature are conflicting. Between early childhood and approximately 8- 9 years of age (i.e. the prepubertal stage), the hypothalamicpituitary- gonadal axis is dormant, as reflected by undetectable serum concentrations of LH and testosterone. In this phase, the activity of the hypothalamus and pituitary may be suppressed by neuronal restraint pathways and by the negative feedback provided in young children by the minute amounts of circulation gonadal steroids. Evidence of hypothalamic-pituitary-gonadal interaction during the prepubertal period resides in the fact that FSH is detectable and may be increased with increasing serum LH.

In cryptorchid boys hCG-treatment induced a marked increase in testosterone into the adult range and completely suppressed FSH and LH. In prepubertal patients aged 4.1-9 years hCG-treatment stimulated inhibin B, whereas no changes or even decreased inhibin B-values were seen in prepubertal patients aged 9-13.6 years (23). The patients did not undergo a testicular biopsy simultaneously with surgery for cryptorchidism (23). However, the different hormonal responses to hCG-treatment in young compared to older prepubertal patients may reflect an age-difference, which may explain why hCG-treatment was not harmful to the germ cells of older compared to younger prepubertal boys. A future research on age-related hormonal dose-response is necessary. The data of our study on germ cells in cryptorchid testes are important in respect of planning a strategy on treatment of cryptorchidism. The operative treatment should be carried out within the first year of life, as long as the germ cell number is not yet severely suppressed (fig. 3), and the natural transformation of germ cells to Ad Spermatogonia seems possible. If the germ cell number is significantly impaired, supplementary hormonal treatment should be considered before a substantial part of the testes lack germ cells, which is approximately at 4 years of age (fig. 1).

Conclusions

At time of birth cryptorchid patients harbour germ cells in the testes, but from about 15 months of age germ cells may lack. The higher age at surgery for cryptorchidism the higher is the risk of finding no germ cells in a testicular biopsy taken at time of surgery. In cases of no germ cells at surgery for cryptorchidism in childhood, there is a very high risk of later infertility. We recommend very early surgery for cryptorchidism, before the germ cells disappear from the testes, which happen from about 15 months of age. We recommend a testicular biopsy simultaneously with surgery for cryptorchidism. The histological findings may be helpful when deciding whether a cryptorchid boy older than 15 months may be offered supplementary hormonal treatment in order to stimulate germ cell proliferation after surgery.

References

Hadziselimovic F and Herzog B., The importance of both an early orchidopexy and germ cell maturation for fertility in cryptorchidism. Lancet. 2001; 358: 1156.